LRRFIP1基因干扰对脂多糖诱导的支气管上皮细胞炎症、氧化应激和细胞凋亡的影响  

The effect of LRRFIP1 gene interference on the inflammation,oxidative stress and apoptosis of bronchial epithelial cells induced by lipopolysaccharide

作　　者：陆芬 汤健[1] LU Fen;TANG Jian(Department of Rehabilitation Medicine,Affiliated Children’s Hospital of Nanjing Medical University,Nanjing 210008,China)

机构地区：[1]南京医科大学附属儿童医院康复医学科,南京210008

出　　处：《免疫学杂志》2020年第10期846-852,共7页Immunological Journal

摘　　要：目的探讨富亮氨酸重复序列相互作用蛋白1(LRRFIP1)基因对脂多糖(LPS)诱导的支气管上皮细胞炎症、氧化应激和细胞凋亡的影响及其作用机制。方法使用LPS处理人支气管上皮细胞株16HBE,并利用Lipofectamine 2000试剂将siLRRFIP1转染到16HBE细胞,实时荧光定量PCR(qRT-PCR)检测LRRFIP1 mRNA表达;免疫印迹试验(Western blot)检测LRRFIP1、细胞周期蛋白D1(CyclinD1)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved caspase-3)、Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤/白血病-2(Bcl-2)、p65、磷酸化p65(p-p65)、IκBα和IκBα和磷酸化IκBα(p-IκBα)蛋白表达;噻唑蓝(MTT)检测细胞活力;试剂盒检测细胞丙二醛(MDA)、超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)水平和炎症因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)和肿瘤坏死因子-α(TNF-α)分泌;流式细胞术检测细胞凋亡。结果LPS诱导的16HBE细胞中LRRFIP1 m RNA、LRRFIP1蛋白表达量、Cleaved caspase-3、Bax、p-p65、p-IκBα蛋白表达量、MDA、LDH、IL-1β、IL-6、TNF-α水平和细胞凋亡率显著提高(P<0.05),CyclinD1、Bcl-2蛋白水平、细胞活力、SOD活性和IL-10水平明显降低(P<0.05),而p65和IκBα蛋白表达量无显著变化。抑制LRRFIP1明显减少LPS诱导的16HBE细胞中LRRFIP1、Cleaved caspase-3、Bax、p-p65、p-IκBα蛋白表达量、MDA、LDH、IL-1β、IL-6、TNF-α水平和细胞凋亡率(P<0.05),并显著增加CyclinD1、Bcl-2蛋白水平、细胞活力、SOD活性和IL-10水平(P<0.05),对p65和IκBα蛋白水平无明显影响。结论抑制LRRFIP1可能通过下调NF-κB信号通路活性及促进脂多糖损伤的支气管上皮细胞的增殖,减轻细胞炎症和氧化应激,并抑制细胞凋亡。This study was performed to investigate the effect of leucine-rich repeat interacting protein 1(LRRFIP1) gene on the inflammation, oxidative stress and apoptosis of bronchial epithelial cells induced by lipopolysaccharide(LPS) and its mechanism. Human bronchial epithelial cell line 16 HBE was treated with LPS, and then transfected with si-LRRFIP1 using Lipofectamine 2000 reagent. qRT-PCR was used to detect LRRFIP1 mRNA expression, while Western blot was employed to detect the expression of LRRFIP1, CyclinD1, Cleaved caspase-3, Bax, Bcl-2, p65, p-p65, IκBα and p-IκBα proteins. MTT was used to determine cell viability;commercial kits were applied to measure the levels of MDA, SOD, LDH and the secretion of inflammatory factors IL-1β, IL-6 and TNF-α;and flow cytometry was employed to examine cell apoptosis. Data showed that in LPS-induced16 HBE cells, the expression of LRRFIP1 mRNA, LRRFIP1 protein, Cleaved caspase-3, Bax, p-p65, p-IκBαproteins, MDA, LDH, IL-1 β, IL-6, TNF-α levels and apoptosis rate were evidently increased, while CyclinD1,Bcl-2 protein levels, cell viability, SOD activity and IL-10 level were markedly reduced, but p65 and IκBα protein expressions did not change significantly. Inhibition of LRRFIP1 obviously decreased the expression of LRRFIP1, Cleaved caspase-3, Bax, p-p65, p-IκBα proteins, MDA, LDH, IL-1β, IL-6, TNF-α levels and apoptosis rate in LPS-induced 16 HBE cells, and dramatically enhanced CyclinD1, Bcl-2 protein levels, cell viability, SOD activity and IL-10 level, while there was no notable effects on p65 and IκBα protein levels. These results indicate that inhibition of LRRFIP1 may promote the proliferation of bronchial epithelial cells damaged by lipopolysaccharide, reduce cell inflammation and oxidative stress, and inhibit apoptosis by down-regulating the activity of NF-κB signaling pathway.

关 键 词：LRRFIP1 支气管上皮细胞 炎症 氧化应激 NF-κB信号通路 

分 类 号：R562.2[医药卫生—呼吸系统]